The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations.

Metformin, an insulin sensitiser from the biguanide family of molecules, is used for the treatment of insulin resistance in type 2 diabetes individuals. It increases peripheral glucose uptake and may reduce food intake. Based on the tight link between metabolism and fertility, we investigated the role of metformin on testicular function using in vitro culture of Sertoli cells and seminiferous tubules, complemented by in vivo data obtained following metformin administration to prepubertal chickens. In vitro, metformin treatment reduced Sertoli cell proliferation without inducing apoptosis and morphological changes. The metabolism of Sertoli cells was affected because lactate secretion by Sertoli cells increased approximately twofold and intracellular free ATP was negatively impacted. Two important pathways regulating proliferation and metabolism in Sertoli cells were assayed. Metformin exposure was not associated with an increased phosphorylation of AKT or ERK. There was a 90% reduction in the proportion of proliferating germ cells after a 96-h exposure of seminiferous tubule cultures to metformin. In vivo, 6-week-old chickens treated with metformin for 3 weeks exhibited reduced testicular weight and a 50% decrease in testosterone levels. The expression of a marker of undifferentiated germ cells was unchanged in contrast to the decrease in expression of 'protamine', a marker of differentiated germ cells. In conclusion, these results suggest that metformin affects the testicular energy content and the proliferative ability of Sertoli and germ cells.

Does uncontrolled cardiac death for organ donation raise ethical questions? An opinion survey.

BACKGROUND: Organ donation after uncontrolled cardiac death raises complex ethical issues. We conducted a survey in a large hospital staff population, including caregivers and administrators, to determine their ethical viewpoints regarding organ donation after uncontrolled cardiac death. METHODS: Multicenter observational survey using a questionnaire, including information on the practical modalities of the procedure. Respondents were asked to answer 15 detailed ethical questions corresponding to different ethical issues raised in the literature. Ethical concerns was defined when respondents expressed ethical concerns in their answers to at least three of nine specifically selected ethical questions. RESULTS: One thousand one hundred ninety-six questionnaires were received, and 1057 could be analysed. According to our definition, 573 respondents out of 1057 (54%) had ethical concerns with regard to donation after cardiac death and 484 (46 %) had no ethical concerns. Physicians (55%) and particularly junior intensivists (65%) tended to have more ethical issues than nurses (52%) and hospital managers (37%). Junior intensivists had more ethical issues than senior intensivists (59%), emergency room physicians (46%) and transplant specialists (43%). CONCLUSION: Only 46% of hospital-based caregivers and managers appear to accept easily the legitimacy of organ donation after cardiac death. A significant number of respondents especially intensivists, expressed concerns over the dilemma between the interests of the individual and those of society. These results underline the need to better inform both healthcare professionals and the general population to help to the development of such procedure.

Identification of BTG2, an antiproliferative p53-dependent component of the DNA damage cellular response pathway.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

The various domains of v-myb and v-ets oncogenes of E26 retrovirus contribute differently, but cooperatively, in transformation of hematopoietic lineages.

The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.

v-erbA oncogene abrogates growth inhibition of chicken embryo fibroblasts induced by retinoic acid.

Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.

Identification of a new gene family specifically expressed in chicken embryonic stem cells and early embryo.

Chicken embryonic stem (CES) cells are pluripotent cells derived from chicken early blastoderm. In order to identify new genes specifically expressed in these pluripotent cells, we have used a gene trap strategy and cloned a novel gene family called cENS for chicken Embryonic Normal Stem cell gene. The cENS genes expression decreases after induction of CES cells differentiation in culture and is restricted in vivo to the very early embryo. We have characterized three different cENS genes. One, cENS-1, is composed of an open reading frame inserted between two terminal direct repeats which are the common point of the cENS genes. cENS-1 encodes a protein identical to cERNI, a recently described protein. cENS-2 is a truncated form of cENS-1. cENS-3 presents two adjacent open reading frames coding respectively for env and pol related proteins. The presence of conserved direct repeats, of retrovirus related genes and the absence of introns argue in favor of a retroviral origin of the cENS genes. In the cENS we identified a promoter region whose activity is strong in CES cells and decreases after induced differentiation showing a highly specific transcriptional activity specific of undifferentiated chicken embryonic stem cells.

Nitrogen budgets for three cropping systems fertilised with cattle manure.

A field plot experiment was set up on a sandy loam soil of SW England in order to determine the efficiency of nitrogen use from different cattle manures. The manure treatments were low and high dry matter cattle slurries and one farmyard manure applied at a target rate of 200 kg total N ha(-1)year(-1), and an untreated control. There were three different cropping systems: ryegrass/clover mixture, maize/rye and maize/bare soil, which were evaluated during 1998/99 and 1999/00. Measurements were made of N losses, N uptake and herbage DM yields. Result showed that manure type had a significant effect on N utilisation only for maize. N balances were negative in maize (approximately -247 to -10 kg N) compared to grass (approximately 5-158 kg N). Agronomic management was more important than manure type in influencing N losses, where soil cultivation appeared to be a key factor when comparing maize and grass systems.

Managing ammonia emissions from livestock production in Europe.

Around 75% of European ammonia (NH(3)) emissions come from livestock production. Emissions occur at all stages of manure management: from buildings housing livestock; during manure storage; following manure application to land; and from urine deposited by livestock on pastures during grazing. Ammoniacal nitrogen (total ammoniacal-nitrogen, TAN) in livestock excreta is the main source of NH(3). At each stage of manure management TAN may be lost, mainly as NH(3), and the remainder passed to the next stage. Hence, measures to reduce NH(3) emissions at the various stages of manure management are interdependent, and the accumulative reduction achieved by combinations of measures is not simply additive. This TAN-flow concept enables rapid and easy estimation of the consequences of NH(3) abatement at one stage of manure management (upstream) on NH(3) emissions at later stages (downstream), and gives unbiased assessment of the most cost-effective measures. We conclude that rapid incorporation of manures into arable land is one of the most cost-effective measures to reduce NH(3) emissions, while covering manure stores and applying slurry by band spreader or injection are more cost-effective than measures to reduce emissions from buildings. These measures are likely to rank highly in most European countries.

Ammonia emissions from cattle, pig and poultry wastes applied to pasture.

In four field experiments, carried out in The Netherlands, small wind-tunnels were used to make direct measurements of ammonia (NH(3)) volatilization from different types of slurry and manure applied to the surface of grassland. During periods of up to six days following application, losses of NH(3)-N often amounted to more than 40% of the NH(4)-N applied. Percentage loss was highest (83%) from a poultry slurry and least (21%) from an air-dried poultry manure. Losses of NH(3)-N were generally greater from pig slurry (36-78%) than from cattle slurry (41%). In most cases 80% or more of the total NH(3)-N loss occurred within 48 h of application. Estimates were made of total annual NH(3) emissions from four systems of poultry housing. The highest total loss (50% of the N voided in droppings) occurred with a battery house producing a slurry with a low content of dry-matter; most of the loss took place after spreading. With a second battery house, in which the droppings were air-dried, the total loss was only 12%, with much lower emissions from the housing and during spreading.

Nutrient run-off following application of livestock wastes to grassland.

Three types of farm waste (cattle slurry, dirty water and farm yard manure (FYM)) were applied to hydrologically isolated grassland plots on a sloping poorly draining soil. Two applications were made, the first in October and the second in February. Application rates were 50 m(3) ha(-1) of slurry and dirty water and 50 t ha(-1) of FYM. Volumes of run-off following rainfall events and concentrations of N, P and K in run-off were measured. Losses of nutrients were higher following applications made with the soil at field capacity and rainfall soon after application. In terms of percentage loss of applied nutrients, losses were generally low. Concentration of N in run-off from the dirty water and FYM treated plots following the first application and the slurry treated plots following the second application exceeded 11.3 mg dm(-3) (a recommended limit for drinking water) although the maximum concentration recorded was 15 mg dm(-3) following FYM application. Concentration of P in run-off only exceeded 1 mg dm(-3) following the second application of cattle slurry. Concentration of K exceeded 10 mg dm(-3) following the first application of FYM and the second application of cattle slurry.

Production of chicken chimeras from injection of frozen-thawed blastodermal cells.

To execute a strategy for reconstituting genetic resources from cryopreserved blastodermal cells, experiments were conducted to optimize conditions for producing chimeric chickens from frozen-thawed blastodermal cells. Stage X blastodermal cells were collected from Barred Plymouth Rock embryos and dispersed. Cells were resuspended in 10% dimethyl sulfoxide in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, and distributed into plastic ampules. Cell suspensions were seeded to induce ice formation at -7 C, cooled from -7 to -35 C at 1 C/min and then ampules were plunged into liquid nitrogen. Thawing was done by plunging the ampules into warm water (37 C) for 3 min. After centrifugation, the supernatant was replaced with DMEM, and dead or broken cells were removed by density gradient centrifugation. Approximately 500 cells were injected into irradiated Stage X White Leghorn recipient embryos. Following incubation, several somatic chimeras were produced. The frequency of somatic chimerism when fresh (unfrozen) cells, or cells that were frozen and selected by density gradient centrifugation on Percoll or Nycoprep were injected into recipient embryos was 84, 79, and 85%, respectively. The percentage of donor-derived pigmentation in the down of these chimeric chickens was 79, 50, and 58%, respectively. Germline chimerism was determined by mating the chimeras that survived to sexual maturity to Barred Plymouth Rocks. Nine of 16 birds (56.2%) injected with fresh cells, 2 of 26 birds (7.7%) injected with cells that were frozen and selected by density gradient centrifugation on a Percoll gradient, and 3 of 26 birds (11.5%) injected with cells that were frozen and selected on a Nycoprep gradient showed germline transmission; the percentage of donor-derived progeny in these chimeras were 29.5, 5.2, and 6.8%, respectively. The Barred Plymouth Rock donor stock was "reconstituted" by inter se mating of germline male and female chimeras. These data demonstrate that the strategy described here for reconstituting genetic resources from cryopreserved blastodermal cells via chimeric intermediates can be performed successfully.

Manipulation of blastodermal cells.

Blastodermal cells isolated from newly laid, unincubated eggs are virtually uncommitted cells that exhibit many of the properties of pluripotential stem cells. They can be transferred from donor to recipient embryos and contribute to both somatic tissues and the germline. Blastodermal cells that have been maintained in culture for 7 d express the epitopes ECMA-7 and SSEA-1, which are also expressed by mouse embryonic stem cells. After culture for up to at least 7 d, blastodermal cells retain the ability to differentiate into somatic tissues and the germline both in vivo and in vitro. Proliferation in the absence of differentiation of blastodermal cells is stimulated by the presence of Leukemia Inhibitory Factor (LIF) and other ligands that interact with the gp130 receptor, and differentiation is stimulated by exposure to retinoic acid. Blastodermal cells also possess high levels of telomerase activity, which is shared by immortalized cells and cells within the germline. Blastodermal cells can be transfected and will express foreign genes both in vivo and in vitro. Transfected cells can be isolated by fluorescence activated cell sorting and can be cryopreserved without losing their ability to contribute to either somatic tissues or the germline. These properties of blastodermal cells make them ideal vectors for introducing genetic modifications to the germline.

The personality factors of the parent of (male and female) schizophrenics.

It was hypothesised that there exists significant difference between the parents of male and of female schizophrenics and those of the corresponding control group in the personality factors. The experimental group consisted of 62 pairs of parents of 62 male and 38 pairs of parents of 38 female schizophrenics. A control group of 100 pairs of parents of mentally and physically healthy off-springs were taken. Subjects were matched for intelligence level, age level, sex, level of education, religion and per capita income and all of them were free from mental illness at any time during their life. The Bengali version of Cattail's 16 PF (C Form) (excluding Factor B) was used. The parents of schizophrenics were found to be significantly different from the corresponding control group in certain personality factors. The results substantiate the hypotheses.

Chicken embryonic stem cells and transgenic strategies.

The production of transgenic birds is an important goal for both fundamental and applied biology. Different methods have been employed to generate transgenic chickens, including microinjection, use of retroviruses and transfection of primordial germ or embryonic germ cells. In this review we will briefly describe these techniques and our efforts to obtain genetically modified avian embryonic stem (ES) cells using liposomes. This latter technique should allow us to modify chicken ES cells with a high efficiency, permitting the rapid generation of transgenic bird lines.

The carbonic anhydrase II gene, a gene regulated by thyroid hormone and erythropoietin, is repressed by the v-erbA oncogene in erythrocytic cells.

The v-erbA oncogene encodes an altered form of the nuclear receptor of the thyroid hormone triiodothyronine (T3). This altered receptor is unable to bind T3, and blocks the differentiation of chicken erythrocyte progenitor cells. To identify the cellular target genes of v-ErbA in the transformed cells, we analysed the expression of several genes in normal erythrocytic cells exposed to T3, and found that the gene encoding carbonic anhydrase II is transcriptionally activated by the hormone. In contrast, this gene is repressed in erythroleukemic cells transformed by the v-erbA product. To investigate in more details the effects of v-ErbA, we constructed a mutant of v-ErbA in which we restored the ability to bind T3. This mutant developed its oncogenicity only in the absence of T3. Upon binding of T3, the transformed cells differentiated and immediately expressed the carbonic anhydrase II gene. These data show that v-ErbA directly inhibits the transcription of the carbonic anhydrase II gene, presumably by competing with normal T3 receptors. The carbonic anhydrase II gene is the first identified target gene of the v-ErbA oncoprotein in erythroleukemic cells.

A novel mechanism of action for v-ErbA: abrogation of the inactivation of transcription factor AP-1 by retinoic acid and thyroid hormone receptors.

Ligand-activated retinoic acid receptor alpha (RAR alpha) and c-ErbA alpha repress the AP-1-mediated transcriptional activation of the interstitial collagenase gene promoter by specifically decreasing the activity of the AP-1 transcription factor. On the other hand, the v-ErbA oncoprotein fails to repress the AP-1 activity and acts as a dominant negative oncoprotein by overcoming the repression of the AP-1 activity induced by RAR alpha and c-ErbA alpha. This maintenance by v-ErbA of a fully active AP-1 complex is correlated with the abrogation by this same oncogene product of the growth-inhibitory response of chicken embryo fibroblasts to retinoic acid treatment. This new mechanism of action of v-ErbA together with its previously discovered dominant repressor effect on transcription of thyroid hormone-activated target genes may explain the contribution of the v-erbA oncogene to sarcomatogenic and leukemogenic transformation.

v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.

Expression of the v-erbA product, an altered nuclear hormone receptor, is sufficient to transform erythrocytic cells in vitro.

We investigated the effect of the v-erbA oncogene product, an altered thyroid hormone receptor, in chicken erythrocyte progenitor cells. Bone marrow cells were infected with a retrovirus vector (XJ12) carrying the v-erbA gene in association with the neoR gene. XJ12-infected erythrocyte progenitor cells gave rise to G418-resistant clones. Some were composed of blast cells identified as transformed CFU-Es blocked in their differentiation. These cells could be grown in culture for at least 25 generations and required anemic chicken serum as a source of erythropoietic growth factors. XJ12 can infect erythrocyte progenitor cells in vivo but is not sufficient to induce erythroleukemia. These data suggest that the activation of a nuclear hormone receptor might represent one step toward the development of neoplasms.

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities.

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185-189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

EGF-R as a hemopoietic growth factor receptor: the c-erbB product is present in chicken erythrocytic progenitors and controls their self-renewal.

c-erbB, encoding the EGF receptor (EGF-R), was originally identified as the cellular homolog of a chicken leukemia oncogene. In humans, EGF-R is distributed widely except in hemopoietic tissues, and its amplification is associated with epidermal and glial malignancies. Here we show that c-erbB is present in normal chicken erythrocytic progenitors and transmits the mitogenic signal induced by TGF alpha. Cells that contain high affinity EGF-R are at approximately the BFU-E stage, and their long-term renewal can be induced by TGF alpha. Upon addition of insulin and erythropoietin, they can be induced to terminally differentiate into red cells. We previously demonstrated that v-erbA blocks differentiation of chicken erythrocytic progenitors but does not abrogate their growth factor dependence for proliferation. These data indicate that proliferation and differentiation are not necessarily coupled in these cells. They also demonstrate a direct role of c-erbB in the control of self-renewal of normal chicken erythrocytic progenitors and could account for the predominant leukemogenic potential of the chicken erbB gene.